| Summary: | 碩士 === 國立臺灣大學 === 生化科技學系 === 99 === The methylotrophic yeast Hansenula polymorpha is one of the most commonly used heterologous protein expression systems. Its methanol oxidase promoter (MOXp) is a strong and tightly regulated promoter, and can be used in heterologous protein expression. Except methanol, the MOXp can also be derepressed by single carbon source starvation such as glycerol, glucose or xylose for expressing downstream genes. Methanol is a commonly used inducer at large-scale fermentation of methylotrophic yeasts in industrial application. The disadvantages of using methanol include that methanol is toxic and flammable, the large-scale production of menthanol would increase the risk, and the cost for managing is high. In addition, high methanol concentration may hurt cells or cause target protein to be degrade. Since therapeutic proteins may face the risk of methanol remanet, it is necessary to develop a methanol-free induction strategy. In this study, using MOXp derepression to express xylanase through different pO2 control modes for regulating glycerol starvation. We found that lower pO2 control can get higher enzyme activity at fixed pO2 control.When pO2 control at 80%, 60% and 40%, the highest xylanase activity are 4368.30±85.65, 6903.73±152.21 and 7512.47±147.13 U/ml (n=3). And the addition of glycerol, base and xylanase activity correlated well with different pO2 control modes, suggesting that the glycerol and base addition can be a good indicator of recombinant protein production in fermentation course.
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